Abstract

Immunotherapy is a promising approach for the treatment of cancers. Modified adenovirus 5 (Ad5) vectors have been used as a platform to deliver genes encoding tumor associated antigens (TAA). A major obstacle to Ad5 vector immunotherapy has been the induction of vector immunity following administration or the presence of pre-existing Ad5 immunity, which results in vector mitigation. It has been reported by us that the Ad5[E1-, E2b-] platform with unique deletions in the E1, E2b and E3 regions can induce potent cell mediated immunity (CMI) against delivered transgene products in the presence of pre-existing Ad5 immunity. Here we report the use of an Ad5[E1-, E2b-] vector platform expressing the TAA HER2/neu as a breast cancer immunotherapeutic agent. Ad5[E1-, E2b-]-HER2/neu induced potent CMI against HER2/neu in Ad5 naïve and Ad5 immune mice. Humoral responses were also induced and antibodies could lyse HER2/neu expressing tumor cells in the presence of complement in vitro. Ad5[E1-, E2b-]-HER2/neu prevented establishment of HER2/neu-expressing tumors and significantly inhibited progression of established tumors in Ad5 naïve and Ad5 immune murine models. These data demonstrate that in vivo delivery of Ad5[E1-, E2b-]-HER2/neu can induce anti-TAA immunity and inhibit progression of HER2/neu expressing cancers.

Ad5 naïve BALB/c mice (n=5/group) were immunized three times using a dose of 108, 109 or 1010 VP Ad5 [E1-, E2b-]-HER2/neu or buffer alone (controls). Fourteen days after the final immunization, splenocytes from the mice were assessed by ELISpot analysis. The greatest induction of CMI was achieved using 1010 VP of the vector. For positive controls, splenocytes were exposed to concanavalin A in all ELISpot assays as described (data not shown). The error bars depict the standard error of the mean (SEM).

Ad5 naïve (A) or Ad5 immune (B) BALB/c mice (n=5/group) were immunized one, two or three times at a seven day intervals with 1010 VP Ad5 [E1-, E2b-]-HER2/neu. Splenocytes were assessed 14 days following the final immunization for IFN-γ and IL-2 secretion by ELISpot assay. No significant CMI responses were observed when splenocytes were exposed to β-galactosidase protein, CMV peptides, or HIV-1 Gag protein (data not shown). Error bars indicate the standard error from the mean.

HER2/neu IgG levels in Ad5 naïve (A) or Ad5 immune (B) mice immunized one (1), two (2), or three (3) times with Ad5 [E1-, E2b-]-HER2/neu. Ad5 naïve or Ad5 immune mice BALB/C mice (n=5) were immunized 3 times at 7-day intervals with 1010 VP Ad5 [E1-E2b]-HER2/neu. Two weeks after the final immunization, serum from the mice was tested by flow cytometry for binding to HER2/neu-expressing cell line CT26-HER2/neu compared to controls. Ad5 naïve sera (C) showed 96.7% reactivity and Ad-immune sera (D) showed 97.8% reactivity (right side peaks). A commercially available antibody to HER2/neu (E) showed reactivity to 95.9% of CT26-HER2/neu cells (right side peaks). Left side peaks represent control serum binding and secondary detection antibody only (shaded). CDCC against CT26-HER2/neu cells was performed (F). Complement mediated lysis was detected in sera from both Ad5 naïve (84.9%) and Ad5 immune (58.7%). No lysis was detected when sera were heat-inactivated. Values represent Mean ± SEM.